Probiotic Properties of Loigolactobacillus coryniformis NA-3 and In Vitro Comparative Evaluation of Live and Heat-Killed Cells for Antioxidant, Anticancer and Immunoregulatory Activities

Some Latiactobacilli are often used as probiotics due to their functional activities, including antioxidant, anticancer and immunoregulation effect. Loigolactobacillus coryniformis NA-3 obtained from our laboratory is a promising probiotic according to the previous study. Coculture, the Oxford cup test and disk-diffusion methods were used to evaluate the probiotic properties and antibiotic resistance of L. coryniformis NA-3. The antioxidant activities of live and heat-killed L. coryniformis NA-3 were assessed via radicals’ scavenging ability. The potential anticancer and immunoregulatory capacity was determined in vitro using cell lines. The results indicate that L. coryniformis NA-3 has antibacterial activity and cholesterol removal ability and is sensitive to most antibiotics. Dead L. coryniformis NA-3 can scavenge free radicals as well as live strains. Live L. coryniformis NA-3 can significantly inhibit the proliferation of colon cancer cells; however, dead cells cannot. After RAW 264.7 macrophages were treated with live and heat-killed L. coryniformis NA-3, the production of NO, IL-6, TNF-α and reactive oxygen species (ROS) was induced. The increased expression of inducible nitric oxide synthase (iNOS) in treated macrophages mediates the production of NO. In conclusion, L. coryniformis NA-3 showed potential probiotic properties, and the heat-killed strain also exhibited activities similar to those of live bacteria, suggesting the possible value of its further application in the food processing and pharmaceutical industries.


Introduction
Lactic acid bacteria (LAB) are a group of lactic-acid-producing, Gram-positive, non-sporeforming, catalase-negative, anaerobic or microaerophilic, cocci or rods [1]. There are many species of LAB, including Enterococcus, Lactobacillus, Pediococcus, Streptococcus, Lactococcus, Vagococcus, Leuconostoc, Oenococcus, Weissella, Carnobacterium and Tetragenococcus, which are general microflora in both human and animal GITs [2]. Some LAB are commonly considered probiotics and generally recognized as safe (GRAS) [3]. Probiotics are defined by the Food and Agriculture Organization (FAO) of the United Nations and the World Health Organization (WHO) as "live microorganisms which when administered in adequate amounts confer a health benefit on the host" [4,5]. Probiotics can confer many benefits such as immunomodulation, lipid and cholesterol reduction, anticancer, antimicrobial and antioxidative properties, prevention of gastrointestinal infections, improvement of lactose metabolism, etc. [6,7]. The functional properties of a probiotic strain can be evaluated via various in vitro tests. The scavenging of free radicals is generally used for the in vitro evaluation of antioxidant activity, and cell experiments are effective methods of estimating biological activities such as anticancer and immunomodulation effects.

Bacteriostatic Circle
The antibacterial ability of Latilactobacillus was measured using its supernatant and the Oxford cup method. L. coryniformis NA-3 was transferred into an 8 mL fresh MRS broth with 1% inoculation and grown for 24 h. The suspension was centrifuged at 10,000 r/min for 5 min. The supernatant was collected and used for the following bactericidal test. The pH of the supernatant was 4.0 after determination. B. cereus, S. Typhimurium, E. coli O157:H7 and S. aureus were also used for bacteriostatic circle tests. MRS medium (original pH 6.0) was used as a blank control. The supernatants of L. coryniformis NA-3 with the pH adjusted to 7.0 and MRS broth with the pH adjusted to 4.0 were also assessed to analyze the antibacterial effect. Petri dishes were placed at room temperature, and bacteriostatic tests were conducted on the following day.

Cholesterol Removal and Bile Salt Hydrolase Production
The Oxford cup test was performed to identify the abilities of cholesterol degradation and BSH production using a Petri dish supplemented with 0.005% cholesterol. One hundred microliters of L. coryniformis NA-3 (10 9 CFU/mL) was added into an Oxford cup, and MRS medium was used as the control. The Petri dish was incubated at 37 • C for 24 h under anaerobic conditions. The appearance of a white precipitating circle indicates cholesterol removal ability and BSH production. The white precipitate was mixed with ninhydrin solution and boiled for 5 min. A color reaction further confirmed the production of BSH.

Preparation of Live and Heat-Killed L. coryniformis NA-3 Cells
One milliliter of L. coryniformis NA-3 was inoculated in 100 mL of MRS medium and grown for 16 h at 37 • C under anaerobic conditions. The bacterial suspension was centrifuged at 12,000 rpm for 5 min. Cells were collected and washed twice with sterile phosphate-buffered saline (PBS, 0.01 M, pH 7.4). Finally, the precipitate was resuspended in sterile PBS and adjusted to 10 8 CFU/mL as a sample of live strains for the next experiment. Half the volume of the bacteria suspension (10 8 CFU/mL) was heat killed at 80 • C for 10 min in a water bath as a sample of heat-killed strains [26].

Observation of Live and Heat-Killed L. coryniformis NA-3 with Scanning Electron Microscope (SEM)
Live and heat-killed L. coryniformis NA-3 were observed using a Hitachi SU8010 scanning electron microscope (Hitachi, Tokyo, Japan) as described in our previous report [25].
2.6. Antioxidant Activity of Live and Heat-Killed L. coryniformis NA-3 2.6.1. ABTS Scavenging The ABTS-radical-scavenging activity assay using live and heat-killed L. coryniformis NA-3 was carried out as described by Song et al. [27], with a few modifications. The mixture of 400 µL of ABTS (Solarbio, Beijing, China) working solution (OD 734 = 0.700 ± 0.02) and 100 µL of live/heat-killed strains was referred to as "Ai". The mixture of 400 µL of ABTS solution (OD 734 = 0.700 ± 0.02) and 100 µL of PBS (10 mM, pH 7.4) was referred to as "Ao". All experiments were performed in triplicate. The mixtures were shocked and left for 6 min in the dark. Two hundred microliters of reaction supernatant was added to a 96-well plate after centrifugation (12,000 rpm for 5 min), and we measured the OD 734 immediately.

DPPH Scavenging
The scavenging activity of live and heat-killed L. coryniformis NA-3 on DPPH (MACK-LIN, Shanghai, China) free radicals was determined as follows [27]. Suspensions of viable and heat-killed L. coryniformis NA-3 (10 8 CFU/mL) were used for assessment, respectively. The mixture of live/heat-killed L. coryniformis NA-3 with DPPH was centrifuged at 12,000 rpm for 5 min, the supernatant of the reaction was added to a 96-well plate, and we measured the OD 517 . PBS was used as the negative control. Each experiment was performed in triplicate.

Nitric Oxide Scavenging
The nitric oxide scavenging activity of the live and heat-killed L. coryniformis NA-3 was measured according to a modified version of the protocol described by Tsai et al. [28]. The supernatant of the reaction was determined at 550 nm. PBS was used as the negative control. Each experiment was performed in triplicate. The scavenging activity (%) = (OD negative control − OD sample )/(OD negative control ) × 100% (3)

Hydroxyl Radical Scavenging
The hydroxyl-radical-scavenging activity of live and heat-killed L. coryniformis NA-3 was evaluated according to our previous protocol, with a few modifications [29]. Live/heatkilled strains were added to the basic reaction system and referred to as "As". PBS was mixed as "Ac". The "Ao" group involved changing H 2 O 2 and the sample to H 2 O and PBS. Each experiment was performed in triplicate. The supernatant of the reaction was added to a 96-well plate, and we immediately measured the optical density at 536 nm.
The scavenging activity (%) = (OD As − OD Ac )/(OD Ao − OD Ac ) × 100% (4) 2.6.5. Superoxide Free Radical Scavenging The superoxide free radical scavenging of live and heat-killed L. coryniformis NA-3 was also determined using the method reported in [29], with a few modifications. The reaction system was prepared as follows: 100 µL of Tris (Solarbio, Beijing, China)-HCl (Beijing Chemical Works, Beijing, China) buffer (pH 8.0, 150 mM), 50 µL of 1,2,3-phentriol (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) (1.50 mM, dissolved with 10 mM HCl) and 200 µL of different samples referred to as "A11". A mixture of Tris-HCl buffer, 10 mM of HCl and different samples was referred to as "A10". A mixture of Tris-HCl buffer, 1,2,3-phentriol and PBS was referred to as "A01". A mixture of Tris-HCl buffer, 10 mM HCl and PBS was referred to as "A00". Each experiment was performed in triplicate. All reactions were vortexed and kept for 30 min in the dark at room temperature. After centrifugation (12,000 rpm for 5 min), 200 µL of supernatant was added to a 96-well plate, and then the OD 325 was determined immediately. HT-29 cells, purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China), were used to assess potential anticancer activity of live and heat-killed L. coryniformis NA-3. The cells were cultured as described in our previous work on macrophages [30]. Fifty thousand cells were seeded in a 96-well plate and grown overnight. HT-29 cells in a 96-well plate were treated with different dosages (10 6 CFU/mL, 10 7 CFU/mL and 10 8 CFU/mL) of live/heat-killed L. coryniformis NA-3 in quadruplicate and cultivated for 24 h. After the cell supernatant was abandoned, the MTS method was used to determine the cell proliferation. The anticancer activity was measured via the proliferation result of HT-29 cells, which was expressed as percentage of OD 490 relative to the negative control.
2.8. The Immunoregulatory Activity of Live and Heat-Killed L. coryniformis NA-3 RAW 264.7 macrophages were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd and cultured using our previous method [30].

Determination of Nitric Oxide
One hundred thousand cells in each well were seeded in a 96-well plate (100 µL/well) and grown for 24 h. A suspension (10 8 CFU/mL) of live/heat-killed strains in PBS was centrifuged and resuspended in DMEM medium, supplemented with 10% FBS, and 1% penicillin-streptomycin, then diluted to 10 7 CFU/mL and 10 6 CFU/mL with DMEM medium containing 10% FBS. Then, the cells were treated with different concentrations of strains. Cells grown in complete DMEM medium were regarded as negative controls. All experiments were performed in quadruplicate.
After 24 h of treatment, the cultured supernatant was collected, and NO production was analyzed via a Griess assay [29]. The absorbance at 550 nm was read using a Synergy H1 microplate reader (BioTek, Winooski, VT, USA). The linear relationship between the concentrations of sodium nitrite and the absorbance was analyzed. The NO production was calculated based on the sodium nitrite standard curve.

Determination of IL-6 and TNF-α
The IL-6 and TNF-α produced by macrophages treated with live/heat-killed L. coryniformis NA-3 was determined by the enzyme-linked immunosorbent assay (ELISA) method. We used ELISA Max™ Deluxe kits (BioLegend, San Diego, CA, USA), according to the manufacturer's instructions.

Proliferation
After collecting cell supernatant (Section 2.8.1), the MTS method was used for proliferation determination as described in Section 2.7.

Intracellular Reactive Oxygen Species Measurement
ROS production in macrophages was quantified by the fluorescence spectrophotometry method described in our previous study [29]. A fluorescence intensity of 485/528 nm in each well was measured using a Synergy H1 microplate reader (BioTek, Winooski, VT, USA). The fluorescence intensity increased with the production of ROS. The result was calculated using the F485/528 result as follows: RAW 264.7 macrophages (500,000 cells/well) were seeded in 6-well plates and grown overnight. The cells were treated with 10 7 CFU/mL of live/ heat-killed L. coryniformis NA-3 and 10 8 CFU/mL of heat-killed L. coryniformis NA-3 for 24 h. Treatment with LPS (1 µg/mL) was regarded as the positive control, and cells grown in DMEM medium with 10% FBS were the negative control.

Western Blot
After being treated for 24 h, the whole cell lysate was extracted, and Western blotting was performed according to our previous study [29]. The band intensity was quantitatively analyzed using ImageJ.

Statistical Analysis
Experiments were conducted at least in triplicate, and the results are presented as mean ± standard deviation. All statistical significances were analyzed using IBM SPSS Statistics Version 26 (IBM, Armonk, NY, USA). The data were evaluated with one-way ANOVA and compared using Duncan's test. p < 0.05 was regarded as statistically significant. Table 2 shows that L. coryniformis NA-3 was resistant to four antibiotics: oxacillin, imipenem, vancomycin and norfloxacin. The judgment standard of minocycline, azithromycin, lincomycin and florfenicol is not referred to at present. Although they had significant inhibition zones, they could not be defined. Another three antibiotics (ceftriaxone, kanamycin and ciprofloxacin) exhibited medium susceptibility for L. coryniformis NA-3. The other 19 antibiotics expressed sensibility for L. coryniformis NA-3. Antibiotic resistance genes can be transferred to other bacteria by conjugating plasmids and transposons [31]. It is hazardous to deliver antibiotic resistance genes to other microorganisms, especially pathogenic bacteria. Moreover, a sensitivity analysis of LAB is important when prescribing probiotic therapy against the background of the use of antibiotics. Our results suggest that L. coryniformis NA- 3 cannot be used for therapy when most antibiotics are used, except for oxacillin, imipenem, vancomycin and norfloxacin. Reports suggest that Latilactobacillus is always sensitive to some antibiotics when used as a probiotic. William P Charteris reported that L. acidophilus, L. casei, L. rhamnosus, L. reuteri and L. fermentum were all sensitive to tetracycline, chloramphenicol, erythromycin and clindamycin when using the disc diffusion method [24]. These are similar to the results for L. coryniformis NA-3. However, Latilactobacillus does not exhibit broad-spectrum susceptibility to antibiotics. After being assessed via the disk diffusion test, L. fermentum, L. crispatus, L. plantarum and L. jensenii were mostly resistant to quinolones and showed moderate sensitivity to some other antibiotics [23]. The properties of L. coryniformis NA-3 are similar to those of Latilactobacillus as described in the literature, i.e., it expresses sensitivity to most antibiotics. The survival rates of B. cereus, S. typhimurium, E. coli O157:H7 and S. aureus after being cocultured with L. coryniformis NA-3 are shown in Figure 1. B. cereus had the highest survival rate of 1.34% at 24 h and 0.31% at 48 h. The survival rate of S. typhimurium (0.35%) was less than that of B. cereus (1.34%) at 24 h. After 48 h, S. typhimurium was undetectable; however, 0.31% of the B. cereus survived. E. coli O157:H7 and S. aureus were almost all dead at 24 h. These results suggest that E. coli O157:H7 and S. aureus were sensitive to L. coryniformis NA-3, and S. typhimurium was also sensitive to it but to a lesser extent. B. cereus exhibited some resistance to L. coryniformis NA-3. The supernatant of L. coryniformis NA-3 also exhibited antibacterial capacity in the four pathogenic bacteria, as Figure 2 shows. Generally, the antibacterial activity of probiotics should be confirmed under the condition of coexist for probiotic and pathogenic bacteria. Coculture is a means of determining the viability of microorganisms grown with other organisms. The results of cocultivation directly suggest that L. coryniformis NA-3 shows antibacterial activity. LAB generally show antibacterial activity, mostly due to their metabolites, such as organic acid (mostly lactic acid), bacteriocin and hydrogen peroxide. The antibacterial ability of metabolites is always evaluated with the supernatant of a bacterial suspension. The bacteriostatic circle method is commonly used to determine the antimicrobial activity of potential probiotics. L. coryniformis NA-3 supernatant showed no effect on the pathogenic bacteria after neutralization with alkali ( Figure 2), and MRS broth supplemented with acid showed an obvious inhibition circle, suggesting that organic acid may be the main antimicrobial substance in the metabolites of L. coryniformis NA-3. The fermentation supernatant of L. plantarum zrx03 isolated from an infant's feces strongly inhibited E. coli JM109, S. aureus and Listeria monocytogenes, and the inhibition zone was significantly observed [32]. The supernatant of L. coryniformis NA-3 acted on B. cereus, S. Typhimurium, E. coli O157:H7 and S. aureus, which showed similar inhibition zones, suggesting good antibacterial activity similar to that of L. plantarum zrx03.

Result of Antibiotic Susceptibility
tabolites is always evaluated with the supernatant of a bacterial suspension. The bacteriostatic circle method is commonly used to determine the antimicrobial activity of potential probiotics. L. coryniformis NA-3 supernatant showed no effect on the pathogenic bacteria after neutralization with alkali ( Figure 2), and MRS broth supplemented with acid showed an obvious inhibition circle, suggesting that organic acid may be the main antimicrobial substance in the metabolites of L. coryniformis NA-3. The fermentation supernatant of L. plantarum zrx03 isolated from an infant's feces strongly inhibited E. coli JM109, S. aureus and Listeria monocytogenes, and the inhibition zone was significantly observed [32]. The supernatant of L. coryniformis NA-3 acted on B. cereus, S. Typhimurium, E. coli O157:H7 and S. aureus, which showed similar inhibition zones, suggesting good antibacterial activity similar to that of L. plantarum zrx03.

Cholesterol Removal and BSH Production Ability
The production of BSH is one of the assessment standards for probiotics. Bile salts can be decomposed into amino acids and cholic acids by BSH. A white precipitate is produced after cholic acids react with cholesterol. Thus, BSH-producing probiotics are helpful for cholesterol removal. White sediment appeared when L. coryniformis NA-3 was grown on a medium supplemented with cholesterol ( Figure 3a). This phenomenon means that a reaction between cholic acid and cholesterol occurred, suggesting the production of BSH. The color reaction of white sediment and ninhydrin was obviously bluish-violet and after neutralization with alkali ( Figure 2), and MRS broth supplemented with acid showed an obvious inhibition circle, suggesting that organic acid may be the main antimicrobial substance in the metabolites of L. coryniformis NA-3. The fermentation supernatant of L. plantarum zrx03 isolated from an infant's feces strongly inhibited E. coli JM109, S. aureus and Listeria monocytogenes, and the inhibition zone was significantly observed [32]. The supernatant of L. coryniformis NA-3 acted on B. cereus, S. Typhimurium, E. coli O157:H7 and S. aureus, which showed similar inhibition zones, suggesting good antibacterial activity similar to that of L. plantarum zrx03.

Cholesterol Removal and BSH Production Ability
The production of BSH is one of the assessment standards for probiotics. Bile salts can be decomposed into amino acids and cholic acids by BSH. A white precipitate is produced after cholic acids react with cholesterol. Thus, BSH-producing probiotics are helpful for cholesterol removal. White sediment appeared when L. coryniformis NA-3 was grown on a medium supplemented with cholesterol ( Figure 3a). This phenomenon means that a reaction between cholic acid and cholesterol occurred, suggesting the production of BSH. The color reaction of white sediment and ninhydrin was obviously bluish-violet and

Cholesterol Removal and BSH Production Ability
The production of BSH is one of the assessment standards for probiotics. Bile salts can be decomposed into amino acids and cholic acids by BSH. A white precipitate is produced after cholic acids react with cholesterol. Thus, BSH-producing probiotics are helpful for cholesterol removal. White sediment appeared when L. coryniformis NA-3 was grown on a medium supplemented with cholesterol ( Figure 3a). This phenomenon means that a reaction between cholic acid and cholesterol occurred, suggesting the production of BSH. The color reaction of white sediment and ninhydrin was obviously bluish-violet and darker than ninhydrin solution and MRS broth (Figure 3b), indicating enzyme production. It has been demonstrated that elevated serum cholesterol levels increase the risk of atherosclerosis and coronary heart disease [33]. Cholesterol removal is always used as an evaluation criterion for probiotics. Bile salt metabolism and cholesterol metabolism are closely related. Deconjugated bile salts by BSH are able to co-precipitate cholesterol at low pH levels, which is an effective in vitro experiment to expose the role of the BSH activity of Latilactobacilli in relation to cholesterol lowering in culture liquid [33]. L. casei ASCC 292 has been reported to exhibit cholesterol removal activity [34]. For L. coryniformis NA-3, similar results to L. casei ASCC 292 were observed. In addition, the ninhydrin reaction is a method for determining the production of enzymes. Results involving white sediment and color reactions suggest the cholesterol removal ability and BSH production of L. coryniformis NA-3.
of Latilactobacilli in relation to cholesterol lowering in culture liquid [33]. L. casei ASCC 292 has been reported to exhibit cholesterol removal activity [34]. For L. coryniformis NA-3, similar results to L. casei ASCC 292 were observed. In addition, the ninhydrin reaction is a method for determining the production of enzymes. Results involving white sediment and color reactions suggest the cholesterol removal ability and BSH production of L. coryniformis NA-3.

Observation of Live and Heat-Killed Strains
The cells of viable and heat-killed L. coryniformis NA-3 were observed with SEM, and the images are shown in Figure 4. L. coryniformis NA-3 was short and rod-shaped, presenting the typical characteristics of lactobacilli. Wirelike objects were attached to the surface of viable L. coryniformis NA-3 (Figure 4a). The attachments were significantly reduced after thermal killing, and they almost disappeared (Figure 4b). However, the integral morphology was not destroyed following heat treatment. Host-microbial interactions are complex molecular mechanisms. When bacteria arrive, host immune cells recognize the conserved microbial components, called "microbial-associated molecular patterns (MAMPs)", through germline-encoded pattern recognition receptors (PRRs), activating innate defense mechanisms [35]. There are some MAMPs on the surface of probiotics, including lipopolysaccharide (LPS), peptidoglycan (PG), lipoteichoic acid (LTA), lipoprotein (LP) and others, which can specifically bind to Toll-like receptors-2 (TLR2) on intestinal cells [35]. As the images show, the cell wall of heat-killed L. coryniformis NA-3 maintained its original dimensional morphology. There were reserved active substances in cell walls, similar to viable bacteria, which may lead to the preservation of some of the biological functions of living L. coryniformis NA-3 cells in heat-killed cells.

Observation of Live and Heat-Killed Strains
The cells of viable and heat-killed L. coryniformis NA-3 were observed with SEM, and the images are shown in Figure 4. L. coryniformis NA-3 was short and rod-shaped, presenting the typical characteristics of lactobacilli. Wirelike objects were attached to the surface of viable L. coryniformis NA-3 (Figure 4a). The attachments were significantly reduced after thermal killing, and they almost disappeared (Figure 4b). However, the integral morphology was not destroyed following heat treatment. Host-microbial interactions are complex molecular mechanisms. When bacteria arrive, host immune cells recognize the conserved microbial components, called "microbial-associated molecular patterns (MAMPs)", through germline-encoded pattern recognition receptors (PRRs), activating innate defense mechanisms [35]. There are some MAMPs on the surface of probiotics, including lipopolysaccharide (LPS), peptidoglycan (PG), lipoteichoic acid (LTA), lipoprotein (LP) and others, which can specifically bind to Toll-like receptors-2 (TLR2) on intestinal cells [35]. As the images show, the cell wall of heat-killed L. coryniformis NA-3 maintained its original dimensional morphology. There were reserved active substances in cell walls, similar to viable bacteria, which may lead to the preservation of some of the biological functions of living L. coryniformis NA-3 cells in heat-killed cells.

Antioxidant Activity
The antioxidant activity of live and heat-killed L. coryniformis NA-3 was evaluated by scavenging free radicals, as Figure 5 shows, including ABTS, DPPH, NO, hydroxyl radical, and superoxide radical. The results suggest that both live and heat-killed L. coryniformis NA-3 exhibited various scavenging capacities for different free radicals. Viable L. coryniformis NA-3 almost did not work on NO; however, heat-killed cells could scavenge a bit of NO (5%), showing better activity than live cells. L. coryniformis NA-3 can significantly scavenge the free radicals of ABTS, DPPH, hydroxyl radical and superoxide radicals. The scavenging ratio of heat-killed L. coryniformis NA-3 (58%) was also about 12% higher than that of viable cells (46%) on ABTS. Untreated L. coryniformis NA-3 was more

Antioxidant Activity
The antioxidant activity of live and heat-killed L. coryniformis NA-3 was evaluated by scavenging free radicals, as Figure 5 shows, including ABTS, DPPH, NO, hydroxyl radical, and superoxide radical. The results suggest that both live and heat-killed L. coryniformis NA-3 exhibited various scavenging capacities for different free radicals. Viable L. coryniformis NA-3 almost did not work on NO; however, heat-killed cells could scavenge a bit of NO (5%), showing better activity than live cells. L. coryniformis NA-3 can significantly scavenge the free radicals of ABTS, DPPH, hydroxyl radical and superoxide radicals. The scavenging ratio of heat-killed L. coryniformis NA-3 (58%) was also about 12% higher than that of viable cells (46%) on ABTS. Untreated L. coryniformis NA-3 was more active than heated cells when scavenging DPPH (45%), hydroxyl radicals (23%) and superoxide radicals (29%). Nonetheless, heat-killed cells also had different effects on scavenging DPPH (32%), hydroxyl radicals (20%) and superoxide radicals (27%), to varying degrees. In conclusion, L. coryniformis NA-3 shows antioxidant activity and can also maintain this capacity after thermal treatment. Heat-killed L. coryniformis NA-3 may be even better than live cells at radical scavenging in some instances.

Antioxidant Activity
The antioxidant activity of live and heat-killed L. coryniformis NA-3 was evaluated by scavenging free radicals, as Figure 5 shows, including ABTS, DPPH, NO, hydroxyl radical, and superoxide radical. The results suggest that both live and heat-killed L. coryniformis NA-3 exhibited various scavenging capacities for different free radicals. Viable L. coryniformis NA-3 almost did not work on NO; however, heat-killed cells could scavenge a bit of NO (5%), showing better activity than live cells. L. coryniformis NA-3 can significantly scavenge the free radicals of ABTS, DPPH, hydroxyl radical and superoxide radicals. The scavenging ratio of heat-killed L. coryniformis NA-3 (58%) was also about 12% higher than that of viable cells (46%) on ABTS. Untreated L. coryniformis NA-3 was more active than heated cells when scavenging DPPH (45%), hydroxyl radicals (23%) and superoxide radicals (29%). Nonetheless, heat-killed cells also had different effects on scavenging DPPH (32%), hydroxyl radicals (20%) and superoxide radicals (27%), to varying degrees. In conclusion, L. coryniformis NA-3 shows antioxidant activity and can also maintain this capacity after thermal treatment. Heat-killed L. coryniformis NA-3 may be even better than live cells at radical scavenging in some instances. It is known that antioxidative compounds on the cell surface are mostly proteins, exopolysaccharides and lipoteichoic acid. Antioxidant enzymes, bioactive peptides and manganese ions that exist in LAB cells also influence their antioxidant effects [11]. A previous study found that the intact cells of L. plantarum MA2 possessed a higher radical scavenging capacity than the culture supernatant and cell-free extract [9]. DPPH and ABTS scavenging activities are generally used to evaluate the antioxidant properties of live or killed lactobacillus. Seo-Jin Yang et al. [11] reported that viable L. plantarum 200655 can scavenge 32% of DPPH and 38% of ABTS free radicals. The live L. coryniformis NA-3 used in our study had higher scavenging activity on DPPH (45%) and ABTS (46%) than It is known that antioxidative compounds on the cell surface are mostly proteins, exopolysaccharides and lipoteichoic acid. Antioxidant enzymes, bioactive peptides and manganese ions that exist in LAB cells also influence their antioxidant effects [11]. A previous study found that the intact cells of L. plantarum MA2 possessed a higher radical scavenging capacity than the culture supernatant and cell-free extract [9]. DPPH and ABTS scavenging activities are generally used to evaluate the antioxidant properties of live or killed lactobacillus. Seo-Jin Yang et al. [11] reported that viable L. plantarum 200655 can scavenge 32% of DPPH and 38% of ABTS free radicals. The live L. coryniformis NA-3 used in our study had higher scavenging activity on DPPH (45%) and ABTS (46%) than the reported L. plantarum 200655. Additionally, live/heat-killed L. plantarum Ln1 from Kimchi and L. plantarum KCTC 3108 were also effective at scavenging DPPH and ABTS [13]. For DPPH, L. coryniformis NA-3 (both live and heat-killed cells) showed higher scavenging activity than Ln1 and KCTC 3108. L. coryniformis NA-3 was weaker than Ln1 and KCTC 3108 at ABTS scavenging. In contrast, heat-killed L. coryniformis NA-3 was significantly better than live L. coryniformis NA-3 when it came to ABTS scavenging, which is different from the reported Ln1 and KCTC 3108. Additionally, our results also suggest that live and heat-killed L. coryniformis NA-3 exhibited NO, hydroxyl radical and superoxide free radical scavenging activities and that heat-killed cells in particular were more effective than live L. coryniformis NA-3 when it came to NO scavenging.

Anticancer Activity
The anticancer activity of live and heat-killed L. coryniformis NA-3 was assessed using colon cancer cell HT-29 ( Figure 6). The proliferation of HT-29 cells treated with heat-inactivated L. coryniformis NA-3 was not significantly different from the negative control, indicating no effect on the growth of cancer cells. Live L. coryniformis NA-3 cells obviously inhibited the proliferation of HT-29 cells with dose dependency. Cells grown with 10 6 CFU/mL L. coryniformis NA-3 were all ineffective at the inhibition of HT-29 cell growth. The proliferation of cancer cells treated with 10 7 CFU/mL (85%) live L. coryniformis NA-3 was significantly decreased when compared with untreated HT-29 cells (100%). HT-29 cells almost could not grow at all when cultivated with 10 8 CFU/mL of live L. coryniformis NA-3. These results indicate that inactive L. coryniformis NA-3 has no inhibitory activity on the growth of cancer cells; however, live bacteria are effective. In vitro studies have demonstrated that live cells of probiotics possess higher antimutagenic activity by improving the host's immune response, altering the metabolic activity of the intestinal microbiota, binding and degrading carcinogens, producing antimutagenic compounds and altering the physiochemical conditions in the colon [36]. Our results revealed that live L. coryniformis NA-3 was effective in terms of its anticancer effects, which may be attributed to some metabolites secreted from the viable strains. The reason that heat-killed L. coryniformis NA-3 cells were not effective in terms of cancer cell proliferation may be their inability to produce anticancer metabolites. It is reasonable to suppose that the metabolites of L. coryniformis NA-3 play an important role in its anticancer activity. However, multiple factors may be involved.

Anticancer Activity
The anticancer activity of live and heat-killed L. coryniformis NA-3 was assessed using colon cancer cell HT-29 ( Figure 6). The proliferation of HT-29 cells treated with heat-inactivated L. coryniformis NA-3 was not significantly different from the negative control, indicating no effect on the growth of cancer cells. Live L. coryniformis NA-3 cells obviously inhibited the proliferation of HT-29 cells with dose dependency. Cells grown with 10 6 CFU/mL L. coryniformis NA-3 were all ineffective at the inhibition of HT-29 cell growth. The proliferation of cancer cells treated with 10 7 CFU/mL (85%) live L. coryniformis NA-3 was significantly decreased when compared with untreated HT-29 cells (100%). HT-29 cells almost could not grow at all when cultivated with 10 8 CFU/mL of live L. coryniformis NA-3. These results indicate that inactive L. coryniformis NA-3 has no inhibitory activity on the growth of cancer cells; however, live bacteria are effective. In vitro studies have demonstrated that live cells of probiotics possess higher antimutagenic activity by improving the host's immune response, altering the metabolic activity of the intestinal microbiota, binding and degrading carcinogens, producing antimutagenic compounds and altering the physiochemical conditions in the colon [36]. Our results revealed that live L. coryniformis NA-3 was effective in terms of its anticancer effects, which may be attributed to some metabolites secreted from the viable strains. The reason that heat-killed L. coryniformis NA-3 cells were not effective in terms of cancer cell proliferation may be their inability to produce anticancer metabolites. It is reasonable to suppose that the metabolites of L. coryniformis NA-3 play an important role in its anticancer activity. However, multiple factors may be involved.  3.6. Immunoregulation Activity 3.6.1. Proliferation A quantitative assay of proliferation was conducted using the MTS method, and the results are exhibited in Figure 7a. Compared to the untreated cells (100%), the proliferation of LPS-treated macrophages was promoted. L. coryniformis NA-3 showed dose-dependent cytotoxicity on macrophages, especially live strains. The proliferation of cells treated with 10 6 CFU/mL of viable and heat-killed L. coryniformis NA-3 showed no significant difference when compared with the negative control (100%), indicating no cytotoxicity. The survivability of macrophages cocultured with 10 7 CFU/mL live and heat-killed L. coryniformis NA-3 was 70% and 72%, showing no significant difference. L. coryniformis NA-3 at 10 8 CFU/mL expressed serious cytotoxicity due to the proliferation of macrophages. The survivability of macrophages treated with 10 8 CFU/mL of viable L. coryniformis NA-3 was only 23%. This value was 43% for the heated samples. Heat-killed L. coryniformis NA-3 had less of an impact than live samples at 10 8 CFU/mL. The proliferation of treatments with 10 7~1 0 8 CFU/mL of samples demonstrated that heat-killed L. coryniformis NA-3 was less detrimental to cell reproduction than live samples and had higher security. A previous report showed that RAW 264.7 cell viability decreased as the concentration of L. rhamnosus GG and L. brevis KCCM 12203P increased. Additionally, viable L. rhamnosus GG and L. brevis KCCM 12203P exhibited lower cell viability than heat-killed cells, which is a similar result to that of our study [27]. security. A previous report showed that RAW 264.7 cell viability decreased as the concen tration of L. rhamnosus GG and L. brevis KCCM 12203P increased. Additionally, viable L rhamnosus GG and L. brevis KCCM 12203P exhibited lower cell viability than heat-killed cells, which is a similar result to that of our study [27].

Production of NO and Cytokines (IL-6 and TNF-α)
A NO assay performed on RAW 264.7 cells was used to evaluate the immune-modulating properties of live and heat-killed L. coryniformis NA-3 in vitro (Figure 7b). Both viable and heat-killed samples can induce NO expression in macrophages. For L. coryniformis NA-3, the untreated and heat-killed cells were insufficient at 10 6 CFU/mL to induce NO production.
When strains were increased to 10 7~1 0 8 CFU/mL, NO was dramatically improved. The treatment groups of live L. coryniformis NA-3 produced 7.2 µM and 13.8 µM of NO at 10 7 CFU/mL and 10 8 CFU/mL, respectively. The production of macrophages induced by 10 7 CFU/mL and 10 8 CFU/mL of heat-killed L. coryniformis NA-3 were 3.4 µM and 12.7 µM NO, respectively, which are less than the values for viable cells. When the dosage of L. coryniformis NA-3 was increased to 10 8 CFU/mL, the different effects of live and heatkilled samples decreased markedly. The data show that heat-treated L. coryniformis NA-3 also had significant immune-regulation properties that are similar to those of viable strains. It has been reported that live lactobacillus possesses immunomodulation characteristics. For example, L. plantarum 200655 isolated from cabbage kimchi enhanced immunity in RAW 264.7 cells by promoting the production of NO and cytokines [10]. Heat-killed L. plantarum emerged as a potentially important modulator of immune response, just as L. plantarum LM1004 enhances early innate immunity by activating macrophages via the TLR-2/MAPK/NF-κB signaling pathway [20]. The moderate release of NO in organisms can cause many physiological effects, such as promoting the activity of natural killer (NK) cells, activating peripheral blood mononuclear cells and regulating T lymphocytes. The excessive or insufficient production of NO produces a series of pathological effects that harm human health, e.g., by destroying DNA and some synthetases. Though NO performs a pivotal role as an immune regulator in a variety of tissues [37], it should be kept within appropriate limits. In this study, we demonstrated that L. coryniformis NA-3 was an effective strain for the NO production of macrophages, suggesting the potential stimulation of immunoregulation. Viable bacteria proliferation may be the main reason causing higher NO production when compared with heat-killed strains. Considering the safety evaluation, heat-killed probiotics may be a better immune activator for applications.
To survey the effect of live and heat-killed L. coryniformis NA-3 on immunoregulation, the production of IL-6 and TNF-α was also evaluated using ELISA. IL-6 and TNF-α were stimulated after cell exposure to live and heat-killed L. coryniformis NA-3. The production of IL-6 ( Figure 7c) in the untreated RAW 264.6 cells was almost 0 pg/mL, whereas the cell supernatant cocultured with 1 µg/mL LPS was measured at 693~768 pg/mL. IL-6 in the cell culture that included heat-treated L. coryniformis NA-3 at 10 7 CFU/mL and live strains at 10 8 CFU/mL was almost undetectable, and no significant differences were detected between them and the negative group (0 pg/mL). However, the stimulation of IL-6 was increased to around 90 pg/mL after being co-induced with 10 7 CFU/mL live samples. Furthermore, IL-6 in cell supernatant treated with heat-killed L. coryniformis NA-3 at 10 8 CFU/mL was approximately 200 pg/mL lower than that of the LPS-stimulated group, which is an acknowledged immunomodulator activator. TNF-α production induced by different samples is illustrated in Figure 7d. Untreated RAW 264.7 macrophages and cells treated with 10 8 CFU/mL of live L. coryniformis NA-3 almost did not secrete TNFα. The production of TNF-α in cell supernatant co-stimulated with 10 7 CFU/mL live L. coryniformis NA-3 was 138 pg/mL, which is obviously more than that observed in the heated strains (61 pg/mL). Heat-treated samples at 10 8 CFU/mL expressed a stronger capacity for TNF-α induction (271 pg/mL) which was even significantly higher than the LPS control (155~190 pg/mL). Above all, both live and heat-killed L. coryniformis NA-3 revealed the stimulation of proinflammatory cytokines, suggesting immunoregulatory activity. Less production of IL-6 and TNF-α in live 10 8 CFU/mL L. coryniformis NA-3 may be mostly due to its toxicity for cell proliferation. IL-6 and TNF-α are two major cytokines secreted by activated macrophages. These results suggest that L. coryniformis NA-3 promotes immunoregulatory effects via stimulating macrophages.
Macrophages are immune cells that play a crucial part in innate and adaptive immune responses. When macrophages are activated, they can produce NO to kill antigens and secrete cytokines such as IL-6 and TNF-α to activate specific immune responses. Many live and heat-killed lactobacilli have been reported to possess immune-enhancing activity by inducing NO and modulatory cytokine profiles in macrophages. For instance, Lactobacillus fermentum DLBSA204 can activate macrophage cells by improving NO and cytokine expression levels [38]. Live and heat-killed L. rhamnosus ATCC 7469 were effective at producing IL-6 and TNF-α in macrophage culture supernatants, suggesting the positive activity of strain L. rhamnosus ATCC 7469 in the modulation or in stimulation of immune responses [39]. The TNF-α production by macrophages cocultured with live L. rhamnosus ATCC 7469 is higher than that in heat-killed cells, which is consistent with L. coryniformis NA-3. IL-6 production is higher in macrophages after treatment with heatkilled L. rhamnosus ATCC 7469 when compared with live cells, which is similar to the result of 10 8 CFU/mL of live and heat-killed L. coryniformis NA-3. NO, IL-6 and TNF-α were also effectively induced when macrophages were treated with both live and heat-inactivated L. casei and L. plantarum cells [40]. For different LAB, live and heat-killed strains show various activities when it comes to inducing NO and cytokines. Our results suggest that live L. coryniformis NA-3 has a better ability to secrete NO and cytokines (IL-6 and TNF-α). However, 10 8 CFU/mL of live L. coryniformis NA-3 greatly inhibited cell proliferation, which is not a beneficial phenomenon. When considering cell cytotoxicity, heat-killed LAB may be safer for further applications.

Reactive Oxygen Species
As Figure 7e shows, intracellular ROS in macrophages could be induced by L. coryniformis NA-3. It exhibited no significant differences in ROS production at 10 6 CFU/mL with untreated cells. However, live L. coryniformis NA-3 expressed a little more potential than the heat-killed treatment. With an increase in dosage, the ROS production of cells treated with 10 7 CFU/mL of live and inviable strains rises, especially for viable bacteria, showing better effect than LPS. At a dose of 10 8 CFU/mL, cells treated with live strains only produce rare ROS, mostly due to cell cytotoxicity. However, 10 8 CFU/mL of inactivated strains could also stimulate ROS production in macrophages that was almost not significantly different from LPS treatment. The ROS production of macrophages treated with live and heat-killed L. coryniformis NA-3 was significantly different. Live L. coryniformis NA-3 can grow and produce metabolites, but heat-killed strains cannot. It is reasonable to speculate that the mechanism for ROS production may be different for live and heat-killed L. coryniformis NA-3. Regardless, both live and heat-inactivated L. coryniformis NA-3 possessed the ability to induce ROS production. High concentration of ROS may lead to non-specific damage to proteins, lipids and nucleic acids, increasing the risk of cardiovascular disease, neurological disorders, cancers and chronic inflammation [41]. Additionally, ROS also play a critical role in various biological functions, including cell survival, cell growth, proliferation and differentiation and immune response [42]. Clearly, 10 6~1 0 8 CFU/mL of live L. coryniformis NA-3 and 10 7~1 0 8 CFU/mL of heat-treated strains have potential immune regulation activity via the inducing of the production of ROS. However, more ROS were generated by macrophages treated with live Latilactobacillus than those treated with inactivated samples at the same dose. In consideration of the risk to health of excessive ROS, heat-killed L. coryniformis NA-3 may be more suitable for use for immunoregulation. Figure 7f shows the result of iNOS expression. Induced nitric oxide synthase (iNOS) expression was examined based on the production of NO (Figure 7a) generated by RAW 264.7 cells. L. coryniformis NA-3 enhanced the iNOS expression in macrophages both with and without heat treatment. Compared to LPS (1 µg/mL), 10 7 CFU/mL of live L. coryniformis NA-3 significantly promoted iNOS production. However, the heat-killed strain was slightly weaker than live L. coryniformis NA-3. Three nitric oxide synthase (NOS) isozymes are responsible for NO production, including endothelial NOS (eNOS), neural NOS and inducible Ca 2+ /NOS (iNOS) [43]. The former two are Ca 2+ -dependent and are expressed constitutively (cNOS). The iNOS expression is Ca 2+ -independent and can provide a high output of NO generation for host defense, once induced [44]. Because NO is induced through the oxidation of L-arginine by iNOS, the production of iNOS cocultured with LAB strains showed similar results to NO production [11]. Previous